HepG2.2.15 cells (obtained from the Cell Center of the Chinese Academy of Medical Sciences; prepared by transfection of HepG2 cells with HBV genome) were maintained in DMEM (HyClone Laboratories, Inc., New England, United States) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin and 380 mg/L G418 in a thermostatic and sealed incubator (37 °C, 5% CO₂). About 100 mmol/L SAHA (Sigma-Aldrich Corp, Missouri, United States) in dimethylsulfoxide (DMSO) was prepared and stored at -20 °C until further use. HepG2.2.15 cells were divided into a control group and several treatment groups to receive different concentrations of SAHA. The adherent cells were washed down with 0.25% trypsin, followed by passage.

An MTT colorimetric assay was used to monitor inhibition of cell proliferation by the addition of different concentrations of SAHA to cell culture medium. For three 96-well plates, 100 μL HepG2.2.15 cells (1 × 10⁵ cells/mL) was added to each well and incubated for 12 h at 37 °C in 5% CO₂. Once cells were adhered to the wells, SAHA was added to a final concentration of 2.5, 5, 7.5 or 10 μmol/L. Wells without SAHA were used as negative controls. After the addition of SAHA, a culture plate was incubated for 24, 48 or 72 h. Cell morphology was examined by confocal laser scanning microscopy (CLSM). Then, 20 μL of MTT (5 mg/mL) was added to each well. After incubation for 4 h, 150 μL of DMSO was added, followed by mixing for 10 min. Lastly, absorbance (A) at 490 nm was measured using a microplate reader. The inhibition rate of cell proliferation was calculated as follows: Cell proliferation inhibition rate (%) = (1 - A_{SAHA group}/A_{Negative control group}) × 100%.

Control group and SAHA groups (2.5 and 5 μmol/L) were cultured for 24 and 48 h respectively. The single cell suspension was then prepared. After centrifugation at 2000 g for 5 min, the cell pellet was resuspended in 0.5 mL PBS (final concentration, 1-5 × 10⁵ cells/mL). For detection of apoptosis, binding buffer (500 μL) and 5 μL annexin V-fluorescein isothiocyanate (Annexin V-FITC) were added to the cell resuspension, followed by 5 μL propidium iodide (PI) (Nanjing KGI Biological Technology Co., Ltd., Nanjing, China). After incubation for 5-15 min (room temperature, avoiding light), samples were subjected to flow cytometry (FCM). For determination of cell cycle phase, 5 mL of obtained cell resuspension was infused into 70% cold ethanol, followed by fixation at 4 °C overnight. During the next day, the cell solution was centrifuged at 800 r/min for 15 min, followed by two phosphate buffer saline (PBS) washes and resuspension in 0.4 mL PBS. RNaseA was added to a final concentration of 50 μg/mL, followed by digestion for 30 min in a 37 °C water bath. Lastly, PI was added to a final concentration of 65 μg/mL, followed by incubation for 30 min. After filtration through a nylon mesh, FCM was conducted.

HepG2.2.15 cells (2.5 × 10⁵ cells/mL) were plated onto a 6-well plate. In triplicate, 1 μL SAHA (7.5 μmol/L) or an equivalent volume of DMSO was added to an individual well. After incubation for 72 h, cells were centrifuged at 800 r/min for 5 min. The supernatant was collected, and the hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) content were quantitated using quantitative chemiluminescence detection kits in i4000sR automatic chemiluminescence immunoassay analyser (R.D. Abbott Company, Inc., California, United States).

A 6-well cell culture plate was prepared as previously described above. HBV negative and positive controls were prepared as follows: 100 μL of cell supernatant was mixed with an equal amount of DNA extraction liquid (shaking for 15 s), followed by centrifugation at 12000 g for 10 min to remove supernatant. Then, 20 μL of DNA extraction liquid was added to the sediment, followed by incubation for 10 min in a 100 °C water bath.

HBV-polymerase chain reaction (PCR) reaction liquid (35.6 μL) and Taq enzyme (0.4 μL) were mixed in a 0.2 mL Eppendorf tube. Two μL of treated sample supernatant was then added to each tube and centrifuged at 8000 g for several seconds. Quantitative fluorescent PCR was performed under the following conditions: 95 °C for 3 min; 94 °C for 15 s (40 cycles); 60 °C for 30 s (40 cycles).

Data were expressed as mean ± SD. Statistical analysis was performed using SPSS 16.0 statistical software. Single factor analysis of variance and t tests were conducted for comparison among multiple groups. P < 0.05 was considered as statistically significant.

CLSM showed that proliferation of untreated HepG2.2.15 cells was rapid, and the cells were compactly arranged. The adjacent cells fused into pieces, with clear edge. The cytoplasm was small, with a large nucleus. In SAHA-treated groups, cell proliferation rate was significantly slower. There were sparse adherent cells, with blurred configuration. The cytoplasm increased, presenting morphological changes similar to normal cells (Figure 1).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Cell morphology changes in confocal laser scanning microscopy. A: Control group; B: Suberoylanilide hydroxamic acid group.

Multiple concentrations of SAHA could inhibit proliferation of HepG2.2.15 cells, and the inhibitory rate increased with increasing concentrations of SAHA (P < 0.05). With each SAHA concentration, the inhibition rate gradually increased with prolonged treatment time (P < 0.05). Taken together, the inhibitory effect of SAHA on cell proliferation was time- and dose-dependent (Table 1).

After 24 h of treatment with 2.5 μmol/L SAHA, early apoptosis rate of HepG2.2.15 cells increased from 3.25% to 16.28% (P = 0.032), and the middle-late apoptosis rate increased from 1.08% to 5.16% (P = 0.035). In the 5 μmol/L SAHA group, early and middle-late apoptosis rate increased from 3.25% to 21.02% (P = 0.041) and 1.08% to 10.70% (P = 0.045), respectively (Table 2 and Figure 2). After 24 h of treatment with 2.5 and 5 μmoL/L SAHA, the proportion of G₀/G₁ phase cells increased from 50.3% to 69.9% and 65.3%, respectively, and the proportion of S phase cells decreased from 34.9% to 22.3% and 20.6%, respectively. Most cells were arrested in the G₀/G₁ phase (Table 3).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Effect of suberoylanilide hydroxamic acid on apoptosis of HepG2. 2.15 cells. A: Control group; B: 2.5 μmol/L suberoylanilide hydroxamic acid (SAHA) group; C: 5.0 μmol/L SAHA group. PI: Propidium iodide.

Positive expression of HBsAg and HBeAg in the SAHA group and control group, respectively, was observed. After 48 h of treatment with SAHA, HBsAg and HBeAg content increased from 12.33 ± 0.62 to 25.42 ± 2.67 (P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85 (P = 0.026), respectively, and HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59 (P = 0.029).

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Its occurrence is related to the multiple-step development process of different genetic alterations. At present, there is no effective treatment method. Suberoylanilide hydroxamic acid (SAHA) is a newly discovered anti-tumor drug which has broad application prospect. It exhibits inhibitory effect of tumor growth, which is been further confirmed in clinical trials.

Histone deacetylase inhibitors (HDACIs) are a class of new anticancer drugs emerging in recent years, which has attracted widespread attention. Previous clinical trials find that, SAHA has broad-spectrum anti-hematological and solid tumor activities, with good tolerance. However, the effect of SAHA on hepatitis B virus (HBV) replication has not been reported.

SAHA inhibits HepG2.2.15 cell proliferation, promotes apoptosis, and stimulates HBV replication. In combination with anti-HBV drugs, SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.

SAHA has been applied in previous clinical trials. Results show that, it has broad-spectrum anti-hematological and solid tumor activities. SAHA can inhibit HepG2.2.15 cell proliferation, deduce the differentiation, and promote the apoptosis. At the same time, it can stimulate the replication of HBV. Therefore, SAHA should be cautiously used for treatment of HCC, and be combined with anti-HBV drugs if necessary. It can be used in the treatment of HBV-negative HCC patients.

HCC is one of the most common malignant tumors, and HDACIs are a series of new anticancer drugs with a wide scope of application. In this manuscript, the authors investigated the effects of suberoylanilide hydroxamic acid, a potent HDACI on proliferation and apoptosis of a human hepatocellular carcinoma cell line (HepG2.2.15) and HBV replication. The manuscript is very well written.